Expression Of Voltage-gated Na Research Articles

Peripheral Voltage-Gated Cation Channels in Neuropathic Pain and Their Potential as Therapeutic Targets.

The persistence of increased excitability and spontaneous activity in injured peripheral neurons is imperative for the development and persistence of many forms of neuropathic pain. This aberrant activity involves increased activity and/or expression of voltage-gated Na+ and Ca2+ channels and hyperpolarization activated cyclic nucleotide gated (HCN) channels as well as decreased function of K+ channels. Because they display limited central side effects, peripherally restricted Na+ and Ca2+ channel blockers and K+ channel activators offer potential therapeutic approaches to pain management. This review outlines the current status and future therapeutic promise of peripherally acting channel modulators. Selective blockers of Nav1.3, Nav1.7, Nav1.8, Cav3.2, and HCN2 and activators of Kv7.2 abrogate signs of neuropathic pain in animal models. Unfortunately, their performance in the clinic has been disappointing; some substances fail to meet therapeutic end points whereas others produce dose-limiting side effects. Despite this, peripheral voltage-gated cation channels retain their promise as therapeutic targets. The way forward may include (i) further structural refinement of K+ channel activators such as retigabine and ASP0819 to improve selectivity and limit toxicity; use or modification of Na+ channel blockers such as vixotrigine, PF-05089771, A803467, PF-01247324, VX-150 or arachnid toxins such as Tap1a; the use of Ca2+ channel blockers such as TTA-P2, TTA-A2, Z 944, ACT709478, and CNCB-2; (ii) improving methods for assessing “pain” as opposed to nociception in rodent models; (iii) recognizing sex differences in pain etiology; (iv) tailoring of therapeutic approaches to meet the symptoms and etiology of pain in individual patients via quantitative sensory testing and other personalized medicine approaches; (v) targeting genetic and biochemical mechanisms controlling channel expression using anti-NGF antibodies such as tanezumab or re-purposed drugs such as vorinostat, a histone methyltransferase inhibitor used in the management of T-cell lymphoma, or cercosporamide a MNK 1/2 inhibitor used in treatment of rheumatoid arthritis; (vi) combination therapy using drugs that are selective for different channel types or regulatory processes; (vii) directing preclinical validation work toward the use of human or human-derived tissue samples; and (viii) application of molecular biological approaches such as clustered regularly interspaced short palindromic repeats (CRISPR) technology.

ALDH2 deficiency induces atrial fibrillation through dysregulated cardiac sodium channel and mitochondrial bioenergetics: A multi-omics analysis

Point mutation in alcohol dehydrogenase 2 (ALDH2), ALDH2*2 results in decreased catalytic enzyme activity and has been found to be associated with different human pathologies. Whether ALDH2*2 would induce cardiac remodeling and increase the attack of atrial fibrillation (AF) remains poorly understood. The present study evaluated the effect of ALDH2*2 mutation on AF susceptibility and unravelled the underlying mechanisms using a multi-omics approach including whole-genome gene expression and proteomics analysis. The in-vivo electrophysiological study showed an increase in the incidence and reduction in the threshold of AF for the mutant mice heterozygous for ALDH2*2 as compared to the wild type littermates. The microarray analysis revealed a reduction in the retinoic acid signals which was accompanied by a downstream reduction in the expression of voltage-gated Na+ channels (SCN5A). The treatment of an antagonist for retinoic acid receptor resulted in a decrease in SCN5A transcript levels. The integrated analysis of the transcriptome and proteome data showed a dysregulation of fatty acid β-oxidation, adenosine triphosphate synthesis via electron transport chain, and activated oxidative responses in the mitochondria. Oral administration of Coenzyme Q10, an essential co-factor known to meliorate mitochondrial oxidative stress and preserve bioenergetics, conferred a protection against AF attack in the mutant ALDH2*2 mice. The multi-omics approach showed the unique pathophysiology mechanisms of concurrent dysregulated SCN5A channel and mitochondrial bioenergetics in AF. This inspired the development of a personalized therapeutic agent, Coenzyme Q10, to protect against AF attack in humans characterized by ALDH2*2 genotype.

K+ Channels in Primary Afferents and Their Role in Nerve Injury-Induced Pain.

Sensory abnormalities generated by nerve injury, peripheral neuropathy or disease are often expressed as neuropathic pain. This type of pain is frequently resistant to therapeutic intervention and may be intractable. Numerous studies have revealed the importance of enduring increases in primary afferent excitability and persistent spontaneous activity in the onset and maintenance of peripherally induced neuropathic pain. Some of this activity results from modulation, increased activity and /or expression of voltage-gated Na+ channels and hyperpolarization-activated cyclic nucleotide–gated (HCN) channels. K+ channels expressed in dorsal root ganglia (DRG) include delayed rectifiers (Kv1.1, 1.2), A-channels (Kv1.4, 3.3, 3.4, 4.1, 4.2, and 4.3), KCNQ or M-channels (Kv7.2, 7.3, 7.4, and 7.5), ATP-sensitive channels (KIR6.2), Ca2+-activated K+ channels (KCa1.1, 2.1, 2.2, 2.3, and 3.1), Na+-activated K+ channels (KCa4.1 and 4.2) and two pore domain leak channels (K2p; TWIK related channels). Function of all K+ channel types is reduced via a multiplicity of processes leading to altered expression and/or post-translational modification. This also increases excitability of DRG cell bodies and nociceptive free nerve endings, alters axonal conduction and increases neurotransmitter release from primary afferent terminals in the spinal dorsal horn. Correlation of these cellular changes with behavioral studies provides almost indisputable evidence for K+ channel dysfunction in the onset and maintenance of neuropathic pain. This idea is underlined by the observation that selective impairment of just one subtype of DRG K+ channel can produce signs of pain in vivo. Whilst it is established that various mediators, including cytokines and growth factors bring about injury-induced changes in DRG function and excitability, evidence presently available points to a seminal role for interleukin 1β (IL-1β) in control of K+ channel function. Despite the current state of knowledge, attempts to target K+ channels for therapeutic pain management have met with limited success. This situation may change with the advent of personalized medicine. Identification of specific sensory abnormalities and genetic profiling of individual patients may predict therapeutic benefit of K+ channel activators.

Inhibition of Electrical Activity by Retroviral Infection with Kir2.1 Transgenes Disrupts Electrical Differentiation of Motoneurons

Network-driven spontaneous electrical activity in the chicken spinal cord regulates a variety of developmental processes including neuronal differentiation and formation of neuromuscular structures. In this study we have examined the effect of chronic inhibition of spinal cord activity on motoneuron survival and differentiation. Early spinal cord activity in chick embryos was blocked using an avian replication-competent retroviral vector RCASBP (B) carrying the inward rectifier potassium channel Kir2.1. Chicken embryos were infected with one of the following constructs: RCASBP(B), RCASBP(B)-Kir2.1, or RCASBP(B)-GFP. Infection of chicken embryos at E2 resulted in widespread expression of the viral protein marker p27 gag throughout the spinal cord. Electrophysiological recordings revealed the presence of functional Kir2.1 channels in RCASBP(B)-Kir2.1 but not in RCASBP(B)-infected embryos. Kir2.1 expression significantly reduced the generation of spontaneous motor movements in chicken embryos developing in ovo. Suppression of spontaneous electrical activity was not due to a reduction in the number of surviving motoneurons or the number of synapses in hindlimb muscle tissue. Disruption of the normal pattern of activity in chicken embryos resulted in a significant downregulation in the functional expression of large-conductance Ca2+-dependent K+ channels. Reduction of spinal cord activity also generates a significant acceleration in the inactivation rate of A-type K+ currents without any significant change in current density. Kir2.1 expression did not affect the expression of voltage-gated Na+ channels or cell capacitance. These experiments demonstrate that chronic inhibition of chicken spinal cord activity causes a significant change in the electrical properties of developing motoneurons.

T1453 Differential Effects of Acute TNBS Colitis On the Expression of Voltage-Gated Na + Channels in Convergent and Non-Convergent Dorsal Root Ganglion Neurons

pain thresholds and change in pain with heterotopic stimulation did not correlate significantly. Conclusions: In healthy subjects there is a good correlation between a simple suprathreshold visceral or somatic pain stimulus and activation of endogenous visceral pain inhibition. The lack of correlation between sensation and pain thresholds and endogenous visceral pain modulation demonstrates the need for suprathreshold stimulation to effectively activate modulation. This study defines a tool which will be useful in the investigation of endogenous visceral pain modulation in patients with functional and other chronic intestinal pain syndromes.

Functional and Molecular Characterization of Voltage-Gated Sodium Channels in Uteri from Nonpregnant Rats1

We investigated the function and expression of voltage-gated Na(+) channels (VGSC) in the uteri of nonpregnant rats using organ bath techniques, intracellular [Ca(2+)] fluorescence measurements, and RT-PCR. In longitudinally arranged whole-tissue uterine strips, veratridine, a VGSC activator, caused the rapid appearance of phasic contractions of irregular frequency and amplitude. After 50-60 min in the continuous presence of veratridine, rhythmic contractions of very regular frequency and slightly increasing amplitude occurred and were sustained for up to 12 h. Both the early and late components of the contractile response to veratridine were inhibited in a concentration-dependent manner by tetrodotoxin (TTX). In small strips dissected from the uterine longitudinal smooth muscle layer and loaded with Fura-2, veratridine also caused rhythmic contractions, accompanied by transient increases in [Ca(2+)](i), which were abolished by treatment with 0.1 microM TTX. Using end-point and real-time quantitative RT-PCR, we detected the presence of the VGSC alpha subunits Scn2a1, Scn3a, Scn5a, and Scn8a in the cDNA from longitudinal muscle. The mRNAs of the auxiliary beta subunits Scbn1b, Scbn2b, Scbn4b, and traces of Scn3b were also present. These data show for the first time that Scn2a1, Scn3a, Scn5a, and Scn8a, as well as all VGSC beta subunits are expressed in the longitudinal smooth muscle layer of the rat myometrium. In addition, our data show that TTX-sensitive VGSC are able to mediate phasic contractions maintained over long periods of time in the uteri of nonpregnant rats.

IL-6 modulates hyperglycemia-induced changes of Na+ channel Beta-3 subunit expression by Schwann cells.

Our data show the suppressive effect of hyperglycemia on expression of voltage-gated Na+ channel beta3 subunit by Schwann cells as well as the protection of beta3 expression by treatment with IL-6. Our findings have direct relevance for therapy of diabetic neuropathy.

Molecular and functional changes in voltage-dependent na + channels following pilocarpine-induced status epilepticus in rat dentate granule cells

Status epilepticus (S.E.) is known to lead to a large number of changes in the expression of voltage-dependent ion channels and neurotransmitter receptors. In the present study, we examined whether an episode of S.E. induced by pilocarpine in vivo alters functional properties and expression of voltage-gated Na + channels in dentate granule cells (DGCs) of the rat hippocampus. Using patch-clamp recordings in isolated DGCs, we show that the voltage-dependent inactivation curve is significantly shifted toward depolarizing potentials following S.E. (half-maximal inactivation at −43.2±0.6 mV) when compared with control rats (−48.2±0.8 mV, P<0.0001). The voltage-dependent activation curve is significantly shifted to more negative potentials following S.E., with half-maximal activation at −28.6±0.8 mV compared with −25.8±0.9 mV in control animals ( P<0.05). The changes in voltage dependence resulted in an augmented window current due to increased overlap between the activation and inactivation curve. In contrast to Na + channel voltage-dependence, S.E. caused no changes in the kinetics of fast or slow recovery from inactivation. The functional changes were accompanied by altered expression of Na + channel subunits measured by real-time reverse transcription–polymerase chain reaction in dentate gyrus microslices. We investigated expression of the pore-forming α subunits Na v1.1–Na v1.3 and Na v1.5–Na v1.6, in addition to the accessory subunits β 1 and β 2. The Na v1.2 and Na v1.6 subunit as well as the β 1 subunit were persistently down-regulated up to 30 days following S.E. The β 2 subunit was transiently down-regulated on the first and third day following S.E. These results indicate that differential changes in Na + channel subunit expression occur in concert with functional changes. Because coexpression of β subunits is known to robustly shift the voltage dependence of inactivation in a hyperpolarizing direction, we speculate that a down-regulation of β-subunit expression may contribute to the depolarizing shift in the inactivation curve following S.E.

Age-related changes in the distribution of Na(v)1.1 and Na(v)1.2 in rat cerebellum.

Modification of sodium channel availability and behavior is obviously a good candidate for alteration of action potential observed during aging. In the present study, we revealed age-related alterations in the expression of voltage-gated Na+ (Na(v)) channel in rat cerebellum by immunohistochemistry. In the cerebellar cortex of aged rats, Na(v)1.1 immunoreactivity in Purkinje cell bodies was highly increased, whereas granule cells showed lower staining intensity. In the cerebellar nuclei of aged rats, Na(v)1.1 and Na(v)1.2 expression was specifically increased in the cerebellar output neurons, which was confirmed by image analysis. The first demonstration of age-related changes in Na(v) channel expression contributes to our understanding of the mechanisms responsible for alteration in synaptic transmission during aging.

Differentiation of Ionic Currents in CNS Progenitor Cells: Dependence upon Substrate Attachment and Epidermal Growth Factor

Multipotential progenitor cells grown from central nervous system (CNS) tissues in defined media supplemented with epidermal growth factor (EGF), when attached to a suitable substratum, differentiate to express neural and glial histochemical markers and morphologies. To assess the functional characteristics of such cells, expression of voltage-gated Na+and K+currents (INa,IK) was studied by whole-cell patch clamp methods in progenitors raised from postnatal rat forebrain. Undifferentiated cells were acutely dissociated from proliferative “spheres,” and differentiated cells were studied 1–25 days after plating spheres onto polylysine/laminin-treated coverslips.INaandIKwere detected together in 58%,INaalone in 11%, andIKalone in 19% of differentiated cells recorded with K+-containing pipettes. With internal Cs+(to isolateINa),INaup to 45 pA/pF was observed in some cells within 1 day after plating.INaranged up to 150 pA/pF subsequently. Overall, 84% of cells expressedINa, with an average of 38 pA/pF.INahad fast kinetics, as in neurons, but steady-state inactivation curves were strongly negative, resembling those of glialINa. Inward tail currents sensitive to [K+]outwere observed upon repolarization after the 10-ms test pulse with internal Cs+, indicating the expression of K+channels in 82% of cells. In contrast to the substantial currents observed in differentiating cells, little or noINaorIK-tail currents were detected in recordings from cells acutely dissociated from spheres. Thus, in the presence of EGF, ionic currents develop early during differentiation induced by attachment to an appropriate substratum. Cells switched from EGF to basic fibroblast growth factor (bFGF) when plated onto coverslips showed greatly reduced proliferation and developed less neuron-like morphologies than cells plated in the presence of EGF.INawas observed in only 53% of bFGF-treated cells, with an average of 9 pA/pF. Thus, in contrast to reports that bFGF promotes neuronal differentiation in some CNS progenitor populations, our EGF-generated postnatal rat CNS progenitors do not develop neuronal characteristics when switched to medium containing bFGF. Thus, differentiated CNS progenitors can express a mix of neuronal and glial molecular, morphological, and electrophysiological properties that can be modified by culture conditions.